It is well recognized that a stagnation or decline in production of edible seafood, in particular, fish, by the marine fishing industry has occurred on a world wide basis. Since the world""s population increases by approximately 100 million each year, maintenance of the present caloric content of the average diet will require production of an additional 19 million metric tons of seafood per year (United Nations Food and Agriculture Organization, The State of the World Fisheries and Aquaculture, Rome, Italy (1995)). In addition, fish products are becoming increasingly utilized in ways other than just food, for example, production of shells and pearls. To achieve this level of production, aquaculture (the cultivation of marine species) will have to double its production in the next 15 years, and wild populations of marine species must be restored.
Aquatic species includes marine teleost and elasmobranch fishes, fresh water teleost fish, euryhaline fish crustations, molusks and echinoderms. Marine teleost fish live in sea water with a high osmolality of about 1,000 mosm. Freshwater teleost fish normally live in water of less than 50 mosm. Euryhaline fish have the ability to acclimate to either of these environments. Ionic composition and osmolality of fish body fluids are maintained in these vastly different environments through gill, kidney and gastrointestinal tract epithelial cell function.
A major problem in aquaculture is development of methodology to rear marine teleost fish, such as cod, flounder and halibut, under freshwater hatchery conditions. To date, factors critical to the acclimation and survival of marine species to fresh water environments, and the control of these factors, have not been fully elucidated.
Attempts to develop such methodologies have also been complicated by problems with feeding the maturing larval forms of these fish. Development of cod, halibut or flounder species that could be reared in fresh water would be of great potential benefit in this regard. Under controlled fresh water conditions, developing forms of these fish could be raised in the absence of bacterial contamination normally present in seawater, and utilize new fresh water food sources that would potentially improve their survival.
The aquaculture industry utilizes the ability of young fish, e.g., salmon, (also called par) to be raised initially in fresh water and subsequently to be transferred for xe2x80x9cgrowth outxe2x80x9d in salt water pens as a means to produce large numbers of adult fish (young salmon tolerant to seawater are called smolt). Improvements in both the survival and health of fish undergoing the par-smolt transition would be very valuable for aquaculture growers.
Moreover, salmon that are kept in coastal marine xe2x80x9cgrow-outxe2x80x9d pens during the winter are constantly at risk, since both winter storms, as well as exposure to extremely cold seawater, causes fish to freeze and die. These risks are further complicated by the fact that when adult salmon are adapted to salt water they do not readily readapt back to fresh water environment. Hence, lack of understanding of the means to readapt adult salmon from salt to fresh water results in the loss of salmon.
It is apparent, therefore, that there is an immediate need to develop methods of augmenting the survival of fish in fresh water and sea water, both in a natural environment and an aquacultural environment.
The present invention relates to the identification and characterization of a PolyValent Cation-sensing Receptor protein (also referred to herein as the Aquatic polyvalent cation-sensing receptor, Aquatic PVCR, or PVCR) which is present in various tissues of marine species. As defined herein, aquatic species includes various fish (e.g., elasmobranch fish, such as sharks, skates; teleost fish, such as summer and winter flounder, salmon, cod, halibut, lumpfish and trout), crustaceans (e.g., lobster, crab and shrimp), mollusks (e.g., clams, mussels and oysters), lamprey and swordfish.
As described herein, for the first time, a polyvalent cation-sensing receptor protein has been identified in aquatic species, located on the plasma membranes of cells in the gastrointestinal tract, kidney, ovary, lung, brain and heart, and in fish brain, gill, heart, intestines, urinary bladder, rectal gland, kidney tubules, and olfactory lamellae. The widespread distribution of Aquatic PVCR protein on the plasma membranes of epithelial cells, as well as in the brain, indicates the involvement of Aquatic PVCR in modulation of epithelial ion and water transport and endocrine function. Data presented herein demonstrate that the Aquatic PVCR plays a critical role in the acclimation of fish to environments of various salinities. The Aquatic polyvalent cation-sensing receptor allows the successful adaptation of fish, such as flounder, to marine and fresh water environments.
One embodiment of the present invention encompasses Aquatic PVCR proteins expressed in tissues of marine species. Aquatic PVCR proteins have been identified as being present in selected epithelial cells in marine, fresh water and euryhaline fish kidney, intestine, gill, urinary bladder, brain, and olfactory tissue. More specifically, the Aquatic PVCR protein has been identified on the plasma membranes of epithelial cells of fish kidney tubules, especially in the collecting duct (CD), late distal tubule (LDT) and the olfactory lamellae. The present invention is intended to encompass these Aquatic PVCR proteins, their amino acid sequences, and nucleic acid sequences, (DNA or RNA) that encode these Aquatic PVCR proteins. In particular, the claimed invention embodies the amino acid and nucleic acid sequences of PVCRs in dogfish shark, winter and summer flounder, and lumpfish.
In another embodiment of the present invention, methods for regulating salinity tolerance in fish are encompassed. Data presented herein indicate that the Aquatic PVCR is a xe2x80x9cmaster switchxe2x80x9d for both endocrine and kidney regulation of adult fish kidney and intestinal ion and water transport, as well as key developmental processes within the fish embryo. Modulating the expression of the Aquatic polyvalent cation-sensing receptor will activate or inhibit Aquatic PVCR mediated ion transport and endocrine changes that permit fish to adapt to fresh or salt water. Also, increasing or deceasing salinity tolerance in aquatic species can refer to activating the PVCR in the epithelial cells.
For example, methods are provided to increase the salinity tolerance of fish adapted to fresh water environment by activation of the Aquatic PVCR in selected epithelial cells. Methods are also provided to decrease the salinity tolerance of fish adapted to a salt water environment by inhibiting the activity of the Aquatic PVCR in selected epithelial cells. Also, regulation of salinity tolerance, via regulating the activation/inhibition of the Aquatic PVCR, occurs by modulating the ion concentration in the surrounding environment. Such modulation can be done by changing the ion concentration of magnesium, calcium and/or sodium.
In another embodiment of the present invention, methods are provided to identify a substance capable of regulating ionic composition of fish fluids, (e.g., salinity tolerance in fish), and endocrine function, by determining the effect that the substance has on the activation or inhibition of the Aquatic PVCR. As described herein, the nucleic acid sequence encoding an Aquatic PVCR has been determined and recombinant PVCR proteins can be expressed in e.g., oocytes of the frog, Xenopus laevis. The oocyte assay system permits the screening of a large library of compounds that will either activate or inhibit Aquatic PVCR function. Candidate compounds can be further screened in e.g., an in vitro assay system using isolated flounder bladder preparations to measure transepithelial transport of ions important for salinity adaption.
As a result of the work described herein, Aquatic PVCR proteins have been identified and their role in maintaining osmoregulation has been characterized. As a further result of the work described herein, methods are now available to modulate the activation of the Aquatic PVCR, resulting in methods to regulate salinity tolerance in marine and fresh water species of fish and thus, facilitate aquaculture of marine fish. Methods of regulating salinity tolerance also provides the means to develop new species of marine fish that are easily adaptable to fresh water aquaculture. Successful development of new species of marine fish would permit these species to be raised initially in protected fresh water hatcheries and later transferred to marine conditions.
The claimed methods also pertain to method for altering body composition (e.g., tissue composition, or meat/muscle composition) comprising modulating the salinity (e.g., ion concentration) of the surrounding environment. Aspects of body composition that are altered include, but are not limited to: fat content, protein content, weight, thickness, moisture, and taste. For example, the thickness of a filet of fish can be increased by the methods described herein. The altering of body composition occurs by maintaining the aquatic species in low and/or high salinity/ion concentrations.
The claimed methods also related to methods for reducing or essentially eliminating or ridding the fish of parasites, bacteria, and contaminants. Maintaining aquatic species in higher salinity than normal reduces parasites and/or bacteria while maintaining the species in lower salinity reduces contaminants (e.g., antibiotics, hydrocarbons, and/or amines). The species can be maintained in both environments, consecutively, to reduce parasites, bacteria and contaminants.